Alcohol/aldehyde dehydrogenase from Gluconobacter oxydans DSM 4025 FERM BP-3812

ABSTRACT

A homogeneous alcohol/aldehyde dehydrogenase isolated from Gluconobacter oxydans DSM 4025 FERM BP-3812 is disclosed. The enzyme is capable in the presence of an electron acceptor, of catalyzing the conversion of L-sorbose to 2-keto-L-gulonic acid via L-sorbosone. The enzyme has a molecular weight of 130,000-140,000 daltons as determined by gel filtration column chromatography, a pyrroloquinoline quinone prosthetic group, an optimum pH for enzyme activity of 7.0 to 9.0, an optimum temperature for enzyme activity from about 20° C. to 40° C. and an isoelectric point of about 4.4. Also disclosed is a process for producing the enzyme and a process for producing aldehydes, carboxylic acids and ketones, especially 2-keto-L-gulonic acid utilizing the enzyme.

BACKGROUND OF THE INVENTION

It is known that there are enzymes which catalyze the oxidation ofalcohols and aldehydes to aldehydes and carboxylic acids, respectively,and have pyrroloquinoline quinone ("PQQ") as a prosthetic group.

Methanol dehydrogenases, which are members of alcohol dehydrogenases,catalyze not only the oxidation of methanol to aldehyde, but alsoformaldehyde to formic acid and formate (Advances in MicrobialPhysiology, 27, 113-209, 1986). These methanol dehydrogenases oxidize awide range of primary alcohols, such as methanol and ethanol and somealdehydes but most of these enzymes cannot oxidize secondary alcohols.The methanol dehydrogenases derived from Methylobacterium organophilum,Pseudomonas C, Diplococcus PAR, and Rhodopseudomonas acidophile areexamples of dehydrogenases which can catalyze the oxidation of secondaryalcohols. To carry out the oxidation of alcohols and aldehydes, themethanol dehydrogenases use activators, such as methylamine or ammonia.

Quinoprotein alcohol dehydrogenase from Pseudomonas aeruginosa (Biochem.J., 223, 921-924, 1984) and quinohaemprotein alcohol dehydrogenase fromPseudomonas testosteroni (Biochem., J., 234, 611-615, 1986) are otherexamples of alcohol dehydrogenases having PQQ as a prosthetic group. Theformer enzyme is a monomer whose molecular weight is 101,000 andrequires ammonium salts or amines as activators. The latter enzyme is amonomer, whose molecular weight is about 67,000, and contains one heam cgroup in its molecule.

SUMMARY OF THE INVENTION

The present invention relates to a novel alcohol/aldehyde dehydrogenase("AADH"), a process for producing the same and a process for producingaldehydes, carboxylic acids and ketones, especially 2-keto-L-gulconicacid ("2-KGA") utilizing said enzyme.

The AADH provided by the present invention catalyzes the oxidation ofalcohols and aldehydes, and is thus capable of producing thecorresponding oxo group from alcohols, and carboxylic acids fromaldehydes. More particularly, the AADH provided by the present inventioncatalyzes the oxidation of L-sorbose to 2-KGA via L-sorbosone. 2-KGA isan important intermediate for the production of vitamin C.

DETAILED DESCRIPTION OF THE INVENTION

The AADH provided by the present invention oxidizes a wide range ofprimary and secondary alcohols to form a corresponding oxo group, andoxidizes aldehydes to form carboxylic acids. The AADH provided by thepresent invention catalyzes the oxidation of L-sorbose to 2-KGA viaL-sorbosone. 2-KGA is an important intermediate for the production ofvitamin C. As used throughout this specification the term oxo grouprefers to aldehydes and ketones. The corresponding oxo group from aprimary alcohol is an aldehyde. The corresponding oxo group from asecondary alcohol is a ketone.

There have been no reports up to now concerning an AADH as provided bythe present invention. The novel AADH reported herein is clearlydistinct from the previously mentioned methanol dehydrogenases in thefollowing ways: AADH hardly oxidizes methanol, while ethanol is a goodsubstrate; AADH does not require ammonia or methylamine as an activatorand AADH has an isoelectric point about 4.4 while the isoelectric pointof most methanol dehydrogenases is higher than 7.0.

The novel AADH is also distinct from the quinoprotein alcoholdehydrogenase mentioned above, in that, this AADH is not a monomer, butconsists of two subunits and this AADH does not require the use of anyactivator. This AADH also differs from quinohaemprotein alcoholdehydrogenase, because this AADH is not a monomer and does not contain aheam c group.

The invention reported herein is a homogenous protein AADH produced by amicroorganism of the genus Gluconobacter. This AADH is an active enzymecapable of oxidizing an alcohol to the corresponding oxo group, andoxidizing an aldehyde to a carboxylic group, in the presence of anelectron acceptor. This AADH has a molecular weight of 135,000±5,000daltons, and is composed of an alpha and beta subunit and apyrroloquinoline quinone prosthetic group. The alpha subunit has amolecular weight of 64,500±2,000 daltons. The beta subunit has amolecular weight of 62,500±2,000 daltons.

The physio-chemical properties of the purified sample of AADH preparedby Examples mentioned herein are further described as follows:

1) Enzyme activity

The AADH of the present invention catalyzes the oxidation of alcoholsand aldehydes, and is capable of producing aldehydes and ketones fromalcohols, and carboxylic acids from aldehydes in the presence of anelectron acceptor.

This electron acceptor may be any conventional compound which has theability to act as an electron acceptor, such as2,6-dichlorophenolindophenol, ("DCIP"), phenazine methosulphate ("PMS"),Wurster's blue, ferricyanide, coenzyme Q or cytochrome c. However, AADHdoes not utilize oxygen as an electron acceptor.

The enzyme assay was performed at 25° C. by measuring the decrease ofabsorbance at 600 nm of DCIP with a spectrophotometer (UVIKON 810,Kontron K.K.). One unit of the enzyme activity was defined as the amountof the enzyme which catalyzed the reduction of 1 μmole of DCIP perminute. The extinction coefficient of DCIP at pH 8.0 was taken as 15mMol⁻¹. The standard reaction mixture (1.0 ml) contained 0.1 mMol DCIP,1 mMol PMS, 125 mMol L-sorbose, 50 mMol Tris-malate-NaOH buffer (pH8.0), and 3-8 μl of the enzyme solution. A reference cuvette containedall the above components except the substrate.

2) Substrate specificity

The substrate specificity of AADH was determined using the same enzymeassay method as described under section 1) above, except that variousfurther substrates were used instead of L-sorbose. The results of themeasurement are shown in Table 1. A variety of compounds such as primaryalcohols, secondary alcohols, aldehydes and high molecular weightalcohols, such as polyethylene glycols or polyvinyl alcohols, can be thesubstrates for AADH.

3) Optimum pH

The correlation between the reaction rate of AADH and pH was determinedin Tris-malate-NaOH buffer (pH 6.0 to 8.5) and in Tris-HCl buffer (pH9.0) using a variety of substrates as shown in Table 2. Regardless ofthe kind of the substrates, AADH showed the highest activity at a pHrange between 7.0 and 9.0. The optimum pH for enzyme activity is 7.0 to9.0.

4) pH stability

Purified AADH was kept standing in buffers of various pH-values for acertain period at 4° C. as shown in Table 3. The residual activity wasassayed under the standard assay condition as described above undersection 1) above using L-sorbose and L-sorbosone as the substrates. Theresults of the measurements are shown in Table 3. The purified enzymewas relatively stable in alkaline pH's and became unstable withincreased acidity.

5) Heat stability

Purified AADH was treated for 10 minutes at various temperatures in 25mMol Tris-HCl buffer (pH 8.0) containing 0.1 Mol NaCl and 5% sucrose,and then cooled immediately in ice water. The residual activity wasmeasured under the standard assay conditions as described undersection 1) above using a variety of substrates. The results are shown inTable 4. AADH is rather stable at and below 30° C., while unstable above40° C.

6) Optimum Temperature

The enzymatic activities of AADH were measured at temperatures from 10°C. to 50° C. in the reaction system as described under section 1) aboveusing a variety of substrates. The results are shown in Table 5. Thisenzyme showed its optimum activity at temperatures between 20° C. and40° C.

7) Molecular Weight

The molecular weight of purified AADH was determined by gel filtrationcolumn chromatography. The sample was applied on a resin for thepurification of proteins, e.g. Sephacryl S-300HR (Pharmacia) columnequilibrated with 25 mMol Tris-HCl buffer (pH 8.0) containing 0.1 MolNaCl and 5% sucrose. As molecular weight standards, thyroglobulin(670,000 dalton), ferritin (450,000 dalton), catalase (240,000 dalton),aldolase (158,000 dalton), gamma globulin (158,000 dalton), bovine serumalbumin (66,200 dalton), ovalbumin (45,000 dalton), chymotrypsinogen A(25,000 dalton), myoglobin (17,000 dalton), cytochome c (12,500 dalton)and vitamin B₁₂ (1,359 dalton) were used. As a result, the molecularweight of AADH was determined to be 135,000±5,000 dalton. Next, purifiedAADH was treated by sodium dodecyl sulfate (SDS) in the presence ofbeta-mercaptoethanol and analyzed for its molecular structure bySDS-polyacrylamide gel electrophoresis. As molecular weight standards,phosphorylase B (92,500 dalton), bovine serum albumin (66,200 dalton),ovalbumin (45,000 dalton), carbonic anhydrase (31,000 dalton), soybeantrypsin inhibitor (21,500 dalton) and lysozyme (14,400 dalton) wereused. It was shown that the enzyme AADH consists of two subunits. One(alpha subunit) has a molecular weight of 64,500±2,000 dalton and theother (beta subunit) has a molecular weight of 62,500±2,000 dalton.

8) Measurement of the Km (Michaelis constant) values

In the procedure described under section 1), the velocities of oxidizingreactions with varying concentrations of several substrates weremeasured to determine the apparent Michaelis constant (Km). The mixtureof DCIP and PMS was used as electron acceptors. The Km values forL-sorbose and 1-propanol were calculated to be 230 mMol and 2 mMol,respectively.

9) Effect of metal ions

Using the assay procedure described under section 1), the effect ofvarious metal ions on the enzyme activity was examined. The results ofthe measurement are shown in Table 6. Among the ions tested, only Mg²⁺and Ca²⁺ did not affect the AADH activity. The others affected theenzyme activity strongly or moderately. Cu²⁺, Mn²⁺ and Fe³⁺ are stronginhibitors for the enzyme.

10) Effect of inhibitors

Using the assay procedure described under section 1) above, the effectof inhibitors on the enzyme activity was examined. The results are shownin Table 7. Ethylenediamine tetraacetic acid ("EDTA") and ethyleneglycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid ("EGTA")strongly inhibited the enzyme activity.

11) Prosthetic group

The absorption spectrum of purified AADH showed an absorption maximum at280 nm followed by a shoulder at 290 nm as shown in FIG. 1. The secondpeak was detected at 340 nm with a wide shoulder at 380-420 nm. Thisabsorption spectrum strongly suggested that AADH has PQQ as a prostheticgroup.

Purified AADH (4.5 mg) in 100 mMol NaH₂ PO₄ --HCl (pH about 1.0) wasadded to an equal volume of methanol and mixed. The sample was thencentrifuged at 15,000 rpm for 10 minutes to remove the precipitate. Theresulting extract was used for the analysis of the prosthetic group. Theabsorption spectrum of the extract was completely identical with anauthentic sample of PQQ (Mitsubishi Gas Chemical Co.). Furthermore, byhigh pressure liquid chromatography analysis, using a reverse phasecolumn (TSK-ODS 80 TM, Toyo Soda CO.), the extract from AADH showed thesame retention time as that of authentic PQQ.

12) Isoelectric point

The isoelectric point (pI) of AADH was determined. Polyacrylamide gel(4%) containing 8.5 Mol urea, 2% (w/v) a non-ionic detergent, e.g.Nonidet P-40 and 2.4% (w/v) of an Ampholite, a buffer component for thepH-gradient, namely Pharmalyte, pH 2.5-5.0 (Pharmacia), was used forisoelectric focusing. The electrode solutions were 0.01 Moliminodiacetate for the anode and 0.01MN-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer forthe cathode. The isoelectric point of the sample was estimated incomparison with a low pH calibration kit, pH 2.5-5.0, purchased fromPharmacia. As a result, AADH showed a cluster containing a few bandshaving pI points at about 4.4.

13) Purification method

The purification of AADH may, in principle, be effected by anycombination of known purification methods, such as:

fractionation with precipitants, e.g. ammonium sulfate, polyethyleneglycol, etc.

ion exchange chromatography,

adsorption chromatography,

gel-filtration chromatography,

gel electrophoresis,

salting out and dialysis.

The AADH provided by the present invention can be prepared bycultivating an appropriate microorganism, disrupting the cells andisolating and purifying it from cell free extract of disrupted cells,preferably from the cytosol fraction of the microorganism.

The microorganisms used in the present invention includes all strainsbelonging to the genus Gluconobacter which are capable of producing AADHcharacterized hereinbefore. Functional equivalents, subcultures, mutantsand variants of said microorganism that are also capable of producingAADH characterized above can be also used in the present invention. Todetermine whether a microorganism of the genus Gluconobacter, or anyfunctional equivalent, subculture, mutant or variant thereof, is capableof producing the novel AADH characterized above, the microorganism canfirst be cultured under aerobic conditions in nutrient medium describedherein, then the AADH may be isolated and purified by conventionalmeans, and the above-listed thirteen (13) physio-chemical properties ofthe purified sample of AADH can be determined and compared to the AADHcharacterized above.

A preferred strain is a specific Gluconobacter oxydans strain, which hasbeen deposited at the Deutsche Sammlung von Mikroorganismen in Gottingen(Germany) under DSM No. 4025 on Mar. 17, 1987.

Moreover, a subculture of said strain has also been deposited in theAgency of Industrial Science and Technology, Fermentation ResearchInstitute, 1-3, Higashi 1 chome Tsukuba-shi Ibaraki-ken 305, Japan,under the stipulations of the Budapest Treaty under the deposit No.:

Gluconobacter oxydans DSM No. 4025 FERM BP-3812 (date of deposit: Mar.30, 1992).

Furthermore, European Patent Publication No. 0278 447 (4226/081k)discloses the characteristics of this preferred strain of Gluconobacteroxydans. Any microorganism having the identifying characteristics ofGluconobacter oxydans DSM No. 4025 (FERM BP-3812) may be used in thepresent invention as a source of novel AADH having the thirteen (13)physio-chemical properties described above. One may determine whether amicroorganism has the identifying characteristics of the depositedstrain of Gluconobacter DSM No. 4025 (FERM BP-3812), either by assay orby direct comparison with the deposited strain.

The microorganisms may be cultured in an aqueous medium supplementedwith appropriate nutrients under aerobic conditions. The cultivation maybe conducted at pH's between about 4.0 and 9.0, preferably between about6.0 and 8.0. While the cultivation period varies depending upon pH,temperature and nutrient medium used, usually 2 to 5 days will bringabout favorable results. A preferred temperature range for carrying outthe cultivation is from about 13° to 36° C., preferably from about 18°to 33° C.

It is usually required that the culture medium contains such nutrientsas assimilable carbon sources, digestable nitrogen sources and inorganicsubstances, vitamins, trace elements and other growth promoting factors.As assimilable carbon sources, L-sorbose, glycerol, D-glucose,D-mannitol, D-fructose, D-arabitol and the like can be used.

Various organic or inorganic substances may also be used as nitrogensource. Among the preferred organic sources, meat extract, peptone,casein, corn steep liquor, urea, amino acids, nitrates, ammonium saltsand the like can be enumerated. As examples of preferred inorganicsubstances, magnesium sulfate, potassium phosphate, ferrous and ferricchlorides, calcium carbonate and the like may be used.

In the following, an embodiment for the isolation and purification ofAADH from microorganisms after the cultivation of these microorganismsis briefly described.

(1) Microorganism cells are harvested from the fermentation broth bycentrifugation or filtration.

(2) These cells are suspended in a buffer solution and disrupted bymeans of a homogenizer, sonicator or treatment with lysozyme and thelike to give a disrupted solution of cells.

(3) AADH is isolated and purified from a cell free extract of thesedisrupted cells, preferably from the cytosol fraction of themicroorganisms.

The AADH provided by the present invention is useful as a catalyst forconverting alcohols to corresponding oxo groups, such as aldehydes andketones, and for converting aldehydes to carboxylic acids. This reactionwherein alcohols and aldehydes are oxidized, comprises the step oftreating the alcohol or aldehyde by contact with the AADH enzymedescribed herein. This AADH enzyme is provided in either or homogenousform or in a non-homogenous form. When the alcohol aldehyde is treatedby contact with a microorganism of the genus Gluconobacter, capable ofproducing the AADH described herein, or by contact with a cell-freeextract of such microorganism, then the AADH is provided in anon-homogenous form. This AADH is especially useful for the productionof 2-KGA from L-sorbose via L-sorbosone.

These reactions utilizing AADH as a catalyst should be conducted at pHvalues from about 6.0 to about 9.0 in a solvent in the presence of anelectron acceptor. Examples of electron acceptors that may be usedinclude DCIP, PMS, Wurster's blue, ferricyanide, coenzyme Q, cytochromec and the like. Any conventional solvent may be used. The preferredsolvents include Tris-HCl buffer, phosphate buffer and the like.

The reaction temperature is not critical; however, the preferredtemperature range for carrying out the above reaction is from about 10°C. to about 50° C. When the reaction is carried out at pH from 7.0-8.0and a temperature within the range of 20° C. and 40° C., best resultsare obtained.

The concentration of the substrate in a solvent can vary depending onother reaction conditions. A substrate concentration from about 10 g/lto about 100 g/l is preferable. Best results are obtained when theconcentration of the substrate is from about 30 g/l to about 40 g/l.

In the above reaction, AADH may also be used in an immobilized statewith an appropriate carrier. Any means of immobilizing enzymes generallyknown to the art may be used. For instance, the enzyme may be bounddirectly to a membrane, or a resin. When AADH is bound to a resin, theenzyme may be bound directly to resin granules by functional group(s),or the enzyme may be bound to the resin indirectly through bridgingcompounds which have functional group(s). The preferred bridgingcompound is glutaraldehyde.

In addition to the above, the cultures cells are also useful for theproduction of aldehydes and ketones from alcohols and for the productionof carboxylic acids from aldehydes, especially for the production of2-KGA from L-sorbose via L-sorborsone.

The following examples further illustrate the present invention.

EXAMPLE 1 Preparation of AADH

All the operations were performed at 4° C. unless otherwise described.

(1) Cultivation of Gluconobacter oxydans DSM No. 4025 (FERM BP-3812)

(a) Preparation of the medium

A seed culture medium containing L-sorbose 8% (w/v) (separatelysterilized), glycerol 0.05%, MgSO₄.7H₂ O 0.25%, corn steep liquor 1.75%,baker's yeast 5.0%, CaCO₃ 0.5% and urea 0.5% (separately sterilized) (pH7.0 before sterilization) was put into a test tube (5 ml each) andsterilized at 120° C. for 20 minutes.

(b) Inoculation, Incubation

Into this seed culture medium, one loopful of the cells of Gluconobacteroxydans DSM No. 4025 (FERM BP-3812) grown on a slant culture mediumcontaining D-mannitol 5.0% in water, MgSO₄.7H₂ O 0.25%, corn steepliquor 1.75%, baker's yeast 0.25%, CaCO₃ 0.5%, urea 0.5% (separatelysterilized) and agar 2.0%, (pH 7.0 before sterilization) at 27° C. forfour days was inoculated and incubated at 30° C. for 24 hours. Theresulting seed culture (5 ml) was inoculated into 100 ml of the sameseed culture medium as described above in a 500 ml-Erlenmeyer flask andincubated at 30° C. for 24 hours. Further, the resulting seed culture (5ml) was inoculated into 100 ml of the same seed culture medium asdescribed above in a 500 ml-Erlenmeyer flask and incubated at 30° C. for24 hours. 750 ml of the seed culture thus prepared were used for theinoculation of 15 l of a main medium in a 30 l jar fermentor. The mediumcontained L-sorbose 10.0% (sterilized separately), glycerol 0.05%, urea1.6% (sterilized separately), MgSO₄.7H₂ O 0.25% baker's yeast 5.0%,CaCO₃ 1.5% and corn steep liquor 3.0%. The fermentation was carried outat 30° C., 500 rpm for the agitation and 7.5 l/minute for the aeration.After 40 hours fermentation, the culture was harvested by centrifugation(10,000 g, 15 minutes). The cell cake was suspended in 1 l of 25 mMolTris-HCl, pH 7.5, containing 0.9% NaCl, 5 mMol MgCl₂ and 1 mMolphenylmethylsulfanyl fluoride (PMSF). The suspension was centrifuged at500 g for 5 minutes to precipitate down CaCO₃ and other precipitatablemedium ingredients. Then, the cells were collected by centrifugation at10,000 g for 15 minutes. The operation as mentioned above was repeatedagain. As a result, 125 g (wet weight) of the cells of Gluconobacteroxydans DSM No. 4025 (FERM BP-3812) were obtained. The washed cells werefrozen at -20° C. for one week before the next purification step.

(2) Preparation of the cytosol fraction

The cells of Gluconobacter oxydans DSM No. 4025 (FERM BP-3812) (125 g)from the above step (1) were suspended in 100 ml of 25 mMol Tris-HClbuffer, pH 8.0, containing 0.5 mMol PMSF and subjected twice to a(French press) cell disrupter to break the cells (1,500 kg/cm²). Intothis homogenized suspension, 2 ml of 1 mg/ml of the DNA splitting DNaseI (Sigma) and 1 ml of 0.5 Mol MgCl₂ were added, the mixture keptstanding for 15 minutes and centrifuged at 6,000 g for 15 minutes toremove the cell debris. The cell free extract (210 ml) thus obtained wascentrifuged at 100,000 g for 60 minutes. The resulting supernatant wascollected as the cytosol fraction (200 ml).

(3) PEG (MW 6000) treatment (precipitation of the DNA)

The cytosol fraction (200 ml) from step (2) was dialyzed overnightagainst 2 liters of 25 mMol Tris-HCl buffer, pH 8.0, containing 0.25mMol PMSF; then 40 g of PEG 6000 (Nakarai Chemicals Ltd.) and 5 ml of 2NKCl, were added and the mixture stirred for 30 minutes and centrifugedat 14,000 g for 20 minutes. The supernatant was filled up to 400 ml withthe same buffer.

(4) DEAE Toyopearl 650M (weak ion-exchange) column chromatography [firststep]

The supernantant (400 ml) obtained from the above step (3) was appliedto a diethylaminoethyl (DEAE) Toyopearl 650M column (2.5 cm in diameterand 35 cm in length), which had been equilibrated with 25 mMol Tris-HClbuffer, pH 8.0, containing 0.25 mMol PMSF and 5% sucrose. After thecolumn was washed with 600 ml of the same buffer, the enzyme was elutedby a linear gradient of NaCl from 0 to 0.5 Mol in the same buffer (2,000ml). The active fractions were pooled (174 ml) and subjected to the nextstep.

(5) Q-Sepharose (strong-ion exchange) column chromatography [secondstep]

The active fractions in the previous step were applied to a Q-Sepharosecolumn (2.5 cm in diameter and 35 cm in length) which had beenequilibrated with 25 mMol Tris-HCl buffer, pH 8.0, containing 5%sucrose. After the column was washed with the buffer to the baseline,the elution of the enzyme was performed with a linear gradient of 0.25to 0.5 Mol NaCl in the same buffer (2,000 ml). The fractions whichcontained electrophoretically homogenous AADH were combined andconcentrated to 20 ml by ultrafiltration using a PM-30 (AmiconCorporation) membrane.

The summary of the purification steps of AADH is shown in Table 8.

(6) Purity of isolated AADH

For the estimation of the purity of isolated AADH, a polyacrylamide gelelectrophoresis was performed. The sample was applied to 7.5%polyacrylamide gel in Tris-HCl buffer, pH 9.4, according to theprocedure of Davis et al. (Ann. N.Y. Acad. Sci. 121: 404, 1969).Proteins were stained with the protein colourant Coomassie BrilliantBlue R-250. The enzyme activity in the gel was detected by coupling itunder the reduction of nitro blue tetrazolium chloride (Sigma). The gelwas immersed at 30° C. in the dark in a solution containing 50 mMolTris-malate buffer, pH 8.0, 0.01 mMol PQQ, 0.1 mMol PMS, 0.4 mMolnitroblue tetrazolium chloride and 0.25 Mol L-sorbose.

AADH showed the closely spaced three bands by protein staining, and allthe bands had enzyme activity. The appearance of three protein bands onthe gel is due to the dissociation of the prosthetic group, PQQ, fromthe enzyme during electrophoresis.

(7) Identification of reaction product

A reaction mixture containing 50 ml of the purified AADH (1.5 mgprotein), 0.1 ml of 10 mMol PMS, 0.5 ml of 0.4 Mol sodium phosphatebuffer, pH 6.5, 0.25 ml of water and 0.1 ml of 20% solution of thevarious substrates was incubated at 30° C. for 15 hours with gentleshaking. The reaction product was analyzed by thin layer chromatography.Products were identified by the direct comparison with authenticsamples. The results are summarized in Table 9.

EXAMPLE 2 2-KGA production by purified AADH

A reaction mixture containing 0.5 ml of purified AADH (15 mg protein,and prepared according to Example 1), 1 ml of a 20% solution ofL-sorbose, 1 ml of 10 mMol PMS, 5 ml of 0.4M sodium phosphate buffer, pH6.5 and 2.5 ml of water was incubated at 30° C. with gentle shaking. Asa result, 2-KGA was formed with the rate of about 70 mg/hour.

EXAMPLE 3 2-KGA production under a resting cell system

The reaction mixture (10 ml): 0.25 g of the cells of Gluconobacteroxydans DSM No. 4025 (FERM BP-3812) prepared in the same manner asdescribed in step (1) of Example 1, 1 ml of a 20% solution of L-sorbose,1 ml of 10 mMol PMS, 1 ml of a 3% aqueous solution of NaCl, 1 ml of 30mMol PQQ, 0.1 g of CaCO₃ and water, was incubated at 30° C. with gentleshaking. As a result, 2-KGA formation was observed with the rate ofabout 6 mg/hour.

We claim:
 1. A homogenous alcohol/aldehyde dehydrogenase isolated fromGluconobacter oxydans DSM 4025 FERM BP-3812, which dehydrogenase iscapable, in the presence of an electron acceptor, of catalyzing theconversion of L-sorbose to 2-keto-L-gulonic acid via L-sorbosone,wherein said dehydrogenase has a molecular weight from 130,000 daltonsto 140,000 daltons as determined by gel filtration columnchromatography, and is composed of an alpha subunit having a molecularweight from 62,500 daltons to 66,500 daltons, a beta subunit having amolecular weight from 60,500 daltons to 64,500 daltons, and apyrroloquinoline quinone prosthetic group, wherein the optimum pH fordehydrogenase activity is from 7.0 to 9.0, the optimum temperature fordehydrogenase activity is from about 20° C. to about 40° C., and whereinsaid dehydrogenase has an isoelectric point of about 4.4.